Serine proteases are one of the most widely studied classes of enzymes: this is largely due to their well-characterised, widespread and diverse roles in a host of physiological and pathological processes. Many disorders are caused by a dysfunction in the normal exquisite regulation of the activity of these proteolytic enzymes, resulting in abnormal tissue destruction and/or aberrant processing of other proteins and peptides. For example, the activity of the serine protease neutrophil elastase (NE), is normally tightly controlled by a variety of native inhibitors such as alpha1 antitrypsin (AAT), secretory leukocyte protease inhibitor (SLPI) and elafin; however, in chronic inflammation such as that found within the lung in cystic fibrosis (CF), this protease overwhelms the tissues natural defences, and the resulting unchecked activity is implicated in tissue destruction, an impaired immune response and ultimately pulmonary decline. Indeed, NE as a biomarker of infection and inflammation has been shown to correlate with the severity of several other respiratory diseases such as chronic obstructive pulmonary disease (COPD) and bronchiectasis (Mayer-Hamblett et al., 2007; Fujimoto et al., 2005; Tsang et al., 2000).
NE has also been found to be elevated in gingival crevice fluid and therefore has value as a biomarker of periodontal disease (Loos and Tjoa, 2005).
Serine proteases have also been implicated as biomarkers in a variety of cancers. For example, human tissue kallikreins (KLKs) which represent the largest group of serine proteases and of which prostate specific antigen (PSA) is the most well known, have attracted particular attention as biomarkers for the screening, diagnosis, prognosis and monitoring of malignant disease (Paliouras et al., 2007). Other serine proteases implicated in tumour-associated events, such as angiogenesis, invasion and metastasis include urokinase and tissue plasminogen activators.
Currently, active proteases are measured predominantly using either chromogenic or fluorogenic substrates which require technical expertise and expensive instrumentation. In addition, these substrates lack selectivity when used with crude biological samples containing a battery of enzymes with multiple proteolytic and hydrolytic proteases. These assays require samples to be processed, which entails the use of expensive instrumentation.
Other methods of detection include immunoassays which are time and labour intensive and due to their expense, require samples to be batched for analysis resulting in long-term storage of samples and the negative impact of freeze/thaw cycles on protein integrity. In addition these assays only measure total protein and cannot differentiate between active and latent enzymes.
Therefore, due to the lack of a rapid and user-friendly detection system, proteases cannot be assayed in the clinic and are rarely assayed in hospital laboratories.
Indeed, although the importance of NE in the pathogenesis of neutrophilic respiratory disease has been established and there is an understanding that routine NE assessment in the clinic would provide important biochemical information which would assist in patient management, NE is only measured in airway samples as an endpoint in research studies and in clinical trials assessing the efficacy of therapeutic interventions. In these cases samples are transferred to contract research laboratories for processing, storage and subsequent analysis.
Up until the early 1990's, research into the association of NE with periodontal disease included placing strips containing a fluorogenic substrate (Prognostiks, Dentsply), directly into the gingival crevice (Eley and Cox, 1998). Fluorogenic substrates used include MeOSuc-Ala-Ala-Pro-Val-7-amido-trifluoro-methylcoumarin (AFC), which was developed by Enzyme System Products (now MP Biomedicals) for Dentsply. This method was not sufficiently sensitive and was also inconvenient for a patient. Another assay system, again based on the fluorogenic substrate detailed above and impregnated into discs, was not commercially developed (Cox et al., 1990).
Other methods currently available to measure proteases such as NE are in ELISA (Enzyme linked Immuno Sorbent Assay) format. For example, in order to measure NE, a NE-alpha1 antitrypsin complex (AAT or α1PI) (Bender Medsystems and Biovendor) is determined. AAT is the endogeneous inhibitor of NE, and in normal conditions an amount of NE-AAT correlates with an amount of released NE. In neutrophilic diseases however, AAT is overwhelmed by the excessive burden of NE, and free active NE can be measured compared to healthy individuals, where no activity can be detected due to effective inhibition. Measurement of NE-AAT complexes alone would therefore only give an indication of inhibited NE, which, as it is already sequestered, cannot cause damage to the surrounding tissue environment, whether it be the gums in dental disease or the lungs in CF. The important measurement has to be that of the unchecked elastinolytic activity. This is well documented in the literature.
Calbiochem currently offer an active NE ELISA on the market in the form of the Innozyme™ NE Immunocapture activity assay kit. By utilising a monoclonal capture antibody to NE, lack of specificity of the subsequent substrate step is compensated for, as the remainder of the crude sample is removed by washing. The major drawback is that the substrate step requires a minimum incubation period of 4 hours up to 24 hours at 37° C., which makes this assay time consuming and impractical for use, particularly in the clinic or hospital laboratories.
Phosphonate probes have been previously reported to target the serine hydrolase family, and selective probes that specifically target the trypsin-like serine protease based on diphenyl phosphosate (DPP)-derived probes have been developed (Hawthorne et al., Anal. Biochem. 326 (2004) 273-275; Pan et al, Bioorg. Med. Chem. Lett. 16 (2006) 2882-2885). However, alternative probes would be useful to aid a better understanding of the activity of protease enzymes in samples to be tested and assays in this regard.